Resorption-controlled bone implants

ABSTRACT

Biomaterials useful to induce the local growth and/or repair of bone tissue are described. The biomaterials incorporate a resorbable matrix doped with an inhibitor of cell-medicated acidification to control the rate at which the matrix is resorbed in vivo. In embodiments, the matrix is a near ambient CAP cement, and the inhibitor is a proton pump inhibitor, such as a macrolide-based antibiotic, especially bafilomycin.

FIELD OF THE INVENTION

The present invention relates to biomaterials of the type useful inlocalized healing and repair of bone tissue. More particularly, thepresent invention relates to biomaterials comprising a bone compatiblematrix and a dopant effective as a proton pump inhibitor, to modulatethe local bony environment within which the biomaterial is implanted”

BACKGROUND OF INVENTION

A variety of biomaterials have been developed for the clinical purposeof forming new bone at fracture sites, and within voids remaining aftersurgical intervention and following bone grafting. These biomaterialsare formed of a matrix material that can be shaped either before orduring application to fill and protect the bone application site duringthe healing process. Over time, the biomaterial is desirably resorbedand replaced by new bone tissue to complete the healing process.

Calcium phosphate (CAP) cements or self-setting cements have attractedconsiderable attention as possible dental implant materials,bone-replacement materials, and drug-delivery vehicles since 1983, whenthey were first described and patented by Brown and Chow. The setting ofcalcium phosphate cements occurs by an acid-base reaction between powderand liquid starting components, which upon mixing form a paste thateventually hardens at room or body temperature. Numerous CAP powders andliquids can enter into the chemical reaction, and the intermediate andfinal products obtained after setting are known to be biocompatible(bone compatible) and osteoconductive.

At the bone tissue repair site, the CAP matrix provides temporary andprotective structure during the repair process and a surface forrecruitment and attachment of bone remodeling cells which form the bonetissue that ultimately replaces the CAP matrix. For clinicalapplications, the CAP implant should be entirely resorbed and replacedby new bone tissue over the treatment period. The rate of CAP resorptionshould correspond to the rate of new bone formation. However, becausethe bone remodeling rate varies at different anatomical sites, and thetreatment period typically varies depending on the site and nature ofthe bone damage, there is a need for technologies that control the rateat which these implants are resorbed.

Current literature addresses the control of CAP biomaterial degradationonly by fine-tuning the CAP chemistry and phase composition. In U.S.Pat. No. 6,117,456 for instance, Lee et al report the desirability ofcontrolling matrix resorption, and suggest that this can be achieved byaltering such physical parameters as density, porosity, reactants, grainsize distribution, final crystallinity, and crystallization inhibitors.This chemical approach results in CAP materials that are either veryinsoluble but also non-resorbable, or CAPs that are highly soluble andhighly resorbable. None of these CAPs offer any control over theosteoclastic resorption, which is the final and most important factorthat dictates clinical performance of the material.

It is an object of the present invention to provide biomaterials thatare useful for the growth and/or repair of bone tissue in vivo.

It is an object of the present invention to provide biomaterials usefulto control acidification in the bony environment to promote the growthand/or repair of bone tissue in vivo.

It is an object of the present invention to provide biomaterials thatare useful for the localized repair and/or healing of bone tissue invivo that comprise a dopant suitable for controlling the rate of matrixresorption in vivo.

It is another object of the present invention to provide a method forrepairing and/or healing bone, comprising the step of applying to a bonesite at which healing and/or repair is desired, a biomaterial of thepresent invention.

It is a further object of the present invention to provide a process forpreparing a CAP biomaterial, which comprises the step of combining asuitable matrix material with a dopant suitable for controlling the rateof resorption thereof in vivo.

SUMMARY OF THE INVENTION

The present invention provides biomaterials that are useful to promotethe localized growth, repair and/or healing of bone tissue in vivo. Thepresent biomaterials incorporate a bone compatible matrix material thatserves as a carrier for the release of a dopant effective as aninhibitor of cell-mediated acidification within the local boneremodeling environment in which the biomaterial is implanted.

In a general aspect, the present invention provides a biomaterial of thetype suitable, when implanted local bony environment, to promote thelocalized growth and/or repair of bone tissue, the biomaterialcomprising a bone compatible matrix material and an inhibitor ofcell-mediated acidification in an amount effective to reducecell-mediated acidification in the bone remodeling environment in whichthe biomaterial is implanted.

In a preferred embodiment, the present biomaterials incorporate a matrixmaterial of the type resorbed over time by cellular processes in theenvironment within which the biomaterial is applied. In particular, thebiomaterials incorporate, as matrix, a material that is resorbed by theacidic environment that is generated during the bone remodeling processby osteoclasts, activated macrophages, and foreign body giant cells. Thepresent biomaterial further incorporates, as dopant, an inhibitor ofcell-mediated acidification in an amount effective to control the rateat which the matrix is resorbed during the healing and/or repairprocess. With incorporation of the dopant, the rate at which the matrixcompositions are resorbed can be controlled to suit the healing andrepair regimens demanded by the various clinical applications in whichsuch matrix materials are useful.

Thus, in accordance with one of its aspects, the present inventionprovides a biomaterial of the type suitable for use in the growth,healing and/or repair of bone, the biomaterial comprising a matrixmaterial that is resorbable in vivo, and an inhibitor of cell-mediatedacidification in an amount effective to control the rate at which saidmatrix is resorbed in vivo.

In accordance with another of its aspects, the present inventionprovides a method for treating a subject to induce the growth or repairof bone tissue, the method comprising the steps of applying, to the siteat which bone growth or repair is desired, a biomaterial of the presentinvention.

In another of its aspects, the present invention provides a process forpreparing a biomaterial of the type useful to induce the growth and/orrepair of bone tissue, the process comprising the steps of combining aresorbable matrix material and an inhibitor of cell-mediatedacidification in an amount effective to control the rate at which saidmatrix material is resorbed in vivo.

In embodiments of the present invention, the matrix comprises a nearambient calcium phosphate cement. In other embodiments of the presentinvention, the acidification inhibitor is a proton pump inhibitor suchas a macrolide antibiotic, including Bafilomycin.

These and other aspects of the present invention are now described ingreater detail with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Section of a rat femur containing the pre-set CAP cement rod 10days after implantation: (A) Control CAP, (B) CAP modified withBafilomycin A₁. Field width 3.4 mm; Hematoxylin & Eosin (H&E) stain.

FIG. 2. Higher magnification of an area shown in FIG. 1 showingdifferences at the tissue/material interface of: (A) Control CAP, (B)CAP modified with Bafilomycin A₁. Field width 0.55 mm. Hematoxylin &Eosin (H&E) stain.

FIG. 3. TRAP stain of osteoclasts at the tissue/material interface: (A)Control CAP, (B) CAP modified with Bafilomycin A₁. Field width 0.097 mm.

FIG. 4. Section of a rat femur containing the injectable CAP cementpaste 14 days after implantation: (A) Control CAP paste, (B) CAP pastemodified with Bafilomycin A₁. Field width 3.4 mm. Masson's trichromestain.

FIG. 5. Section of rat femur containing the pre-set CAP cement rod 10days after implantation showing the differences in local bone massaround the implant: (A) Control CAP, (B) CAP modified with BafilomycinA₁. Field width 9.1 mm. H&E stain.

FIG. 6. Section of rat femur containing the pre-set CAP cement rod 10days after implantation into aged OVX rats. (A) CAP, (3) CAP modifiedwith Bafilomycin A₁. Masson's Trichrome stain.

FIG. 7. Section of rat femur containing the pre-set CAP cement rod 4months after implantation into aged OVX rats. (A) CAP, (B) CAP modifiedwith Bafilomycin A₁. Masson's Trichrome stain.

FIG. 8. Section of rat femur containing the pre-set CAP cement rod 10days after implantation into young Wistar rats. (A) CAP, (B) CAPmodified with PANTO IV. Masson's Trichrome stain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biomaterials that are useful to inducethe localized growth, repair and/or healing of bone tissue. The presentbiomaterials incorporate a bone compatible matrix material that can beshaped for application to the site at which bone growth, healing and/orrepair is desired. Such sites typically include voids created by defect,by fracture, by surgical removal of bone lesions, by implantation ofgrafted bone, and the like including natural voids such as those in thevertebrae in which bone may be fractured.

In embodiments of the invention, in which the biomaterial is used topromote the growth of bone, the inhibitor of cell mediated acidificationis incorporated with a bone compatible matrix that can either be of thetype resorbable over time, as discussed in greater detail below, or ofthe type that constitutes a substantially permanent, and hencesubstantially non-resorbable, structure. These permanent structurestypically are intended to provide a long-lasting bone filler materialhaving structural integrity sufficient to replace bone over longperiods. Included among such matrices are the plastics, particularlyincluding the acrylic polymers, and especially thepolymethylmethacrylates, or PMMAs.

In other embodiments of the present invention, the matrix is composed ofa material that is resorbed in vivo to allow for its replacement bynatural bone following the natural bone remodeling process.Particularly, the term “resorbable matrix material” refers to materialthat is resorbed when exposed in vivo to the acidic environment that iscreated naturally during the bone remodeling process. This acidicenvironment is created by osteoclasts and, to some extent also byactivated macrophages and possibly foreign body giant cells, that arerecruited to the remodeling site, and results likely from the expulsionof H⁺ from proton pumps active in these cell types.

It will thus be appreciated that resorbable matrix materials do notinclude those bone implant materials that are not biodegraded, orresorbed, but instead are designed for load bearing, structural reasonsto be retained at the implant site over the life span of the recipient.In embodiments, the matrix materials suitable for use in the presentbiomaterials rather are those designed, following application, to beresorbed and replaced by natural bone, over time periods ranginggenerally from several days to many months. This resorption property ofthe matrix material can be determined using any one or more of severalassays of matrix resorption that are now well established in this field,including the assay exemplified herein.

Particularly suitable matrix materials are the biodegradable calciumphosphate (CAP)-based cements and pastes. To the extent they may bemodified to allow for resorption over time, the CAP-based matrices canalso include the CAP ceramics. CAP injectable cements, CAP lithomorphs,as well as combinations of these materials with other agents aresuitable, including the slowly biodegradable CAP material sold under thetrade name Calcibon®. Particularly useful CAP matrix materials typicallycomprise dicalcium phosphate, tricalcium phosphate, tetracalciumphosphate and hydroxyapatite, as well as blends of two or more of these.CAP matrix materials suitable in accordance with the present inventioninclude those having a poorly crystalline hydroxyapatite component,formed with amorphous calcium phosphate (Ca/P of about 1.5) as describedin U.S. Pat. No. 6,117,456, the disclosure of which is incorporatedherein by reference. In one embodiment of the present invention, the CAPmatrix comprises a combination of dicalcium phosphate and tetracalciumphosphate.

Also suitable as resorbable matrix materials are the polymeric matrixcompositions, in either two-dimensional or three-dimensional form, thatare based for example on the polyesters including, which may includepolylactides and polyglycolides and copolymers thereof (the PLGAs). Suchmatrix materials include, for instance, the co-polymer described by Holyet al in Biomaterials, 1999, 20(13):1177-85. This material, alsoreferred to as Osteofoam™, is a macroporous biodegradable andbiocompatible tissue engineering scaffold made ofpoly(lactide-co-glycolide) PLGA 75/25 co-polymer. The macroporousinterconnected pore structure of Osteofoam™ resembles that of trabecularbone and allows tissue invasion throughout the scaffold and not only onthe scaffold outer surface. The chemical makeup of this scaffold allowsfor biodegradation of the scaffold to non-cytotoxic degradationproducts. The polymer is degraded in acidic environments, and thereforecan be degraded in vivo by the acidic environment created by theosteoclasts or macrophages.

The resorbable matrix materials can further be selected from any of thevarious tissue engineering scaffolds that are in development, some ofwhich include organic synthetic polymers as well as naturally occurringpolymers such as fibrin, provided that the resorption of such matrixmaterials is mediated at least in part by theosteoclast/macrophage-mediated acidification of the remodelingenvironment. Also, the matrix material can also comprise thedemineralized bone matrix the use of which is well established in theart.

In the prior art, control over the rate at which the matrix is resorbedat the application site has been achieved by altering thephysico-chemical properties of the matrix. By contrast, the presentinvention involves the use of biological/biochemical agents to controlmatrix resorption. More particularly, in the present biomaterials, thematrix is formulated, or doped, with an inhibitor of cell-mediatedacidification, as a means to reduce the rate at which the matrixmaterial would be resorbed, or otherwise biodegraded through acidicdegradation, at the application site. By incorporating such inhibitors,the longevity of any resorbable matrix can thus be tailored to meet therequirements at the particular treatment site.

When doped with acidification inhibitor in amounts effective to controlthe matrix resorption rate, it has been quite surprisingly beendiscovered that there is net formation of new bone at the implant site.Accordingly, the present biomaterials include those which comprise adose of acidification inhibitor that promotes the growth of new bone atthe implant site. The present invention further embraces biomaterials ofthe present invention, comprising a resorbable bone compatible matrixand an inhibitor of cell-mediated acidification, for use in promotingthe formation of new bone.

The acidification inhibitors have the property of reducing the level atwhich the bone remodeling environment is acidified in vivo during theremodeling process. It is to be understood that suitable acidificationinhibitors are those which act mechanistically to reduce the level orrate at which resorbing cells such as osteoclasts and activatedmacrophages release protons into the remodeling environment, typicallyby affecting the exocytic and/or endocytic membrane transport processesin the osteoclast. The term “acidification inhibitor” is not intended toembrace those agents which act biologically to reduce the number orviability of osteoclasts recruited to the remodeling environment. Moreparticularly, the acidification inhibitors are those compounds or agentsthat act either directly or indirectly to inhibit the biochemicalcascade by which protons are released from the resorbing cells, therebyto reduce the level and/or rate at which the remodeling environmentbecomes acidified.

An assay suitable for identifying such acidification inhibitors isdescribed by Davies et al in Cells and Materials, 1993, 3, 245-256, andis also disclosed in U.S. Pat. Nos. 5,766,669; 5,849,569; and 5,861,306,the disclosures of which are incorporated herein by reference. Briefly,the assay utilizes a two-dimensional surface that has been coated with amatrix material, such as CAP, and on which osteoclasts are then culturedwith or without resorption inhibitors. By this assay, inhibitors ofcell-mediated acidification are identified as those compounds which,relative to an untreated control, have no substantial impact on theviability of the osteoclasts or on the number of attached osteoclasts,but which nevertheless cause a reduction in osteoclast-mediated CAPresorption as revealed by a reduction in the degree to which the matrixcoating is dissolved by the osteoclasts, relative to a negative controlhaving osteoclasts cultured on it in the absence of the acidificationinhibitor). Reagents suitable for performing this assay are nowcommercially available, and are sold for instance by Becton Dickinsonunder the trade name Osteologic™, either as discs or as MultiTestslides.

Most desirably, the selected acidification inhibitor is one that doesnot bind too strongly to the matrix material, or to the bone tissue inthe environment in which it is applied. Suitable inhibitors are thosethat can be liberated in detectable yield from the matrix by washing forinstance in PBS or other solvent suited to the inhibitor. The inhibitoralso should remain active following the doping of the matrix therewith.Thus, suitable inhibitors will remain active in the assay just describedfollowing their extraction from the matrix.

The suitable acidification inhibitors also have the property of allowingthe osteoclasts to adhere to the matrix surface, in order to form theisolated osteoclastic lacunae environment. Inhibitors that result in thedesired osteoclast spreading morphology can be further examined usingthe Osteologic assay to confirm that resorptive pits are formed in thepresence of the compound, but with a volume and number that is reducedrelative to an untreated control. Still further, the lacunae areas underthe plated osteoclasts and the osteoclasts themselves can be marked withan acidotropic dye such as3-(2,4-dinitroanillino)-3′-amino-N-methyldipropylamine (DAMP). Theintensity of the stain, measured for instance by fluorescence, canthereby serve to reveal desired acidification inhibitors as thosecompounds that elicit a reduction in acidification, particularly at theosteoclast ruffled border and the matrix surface under the lacunae. Suchan assay is described for instance by Akisaka et al in Cell Tissue Res.,1999, 298(3):527-37, which is incorporated herein by reference.

Still other assay formats are suitable for identifying inhibitors ofcell-mediated acidification. These include in vitro bioassays in whichbone resorption is assessed in the presence and absence of the candidateacidification inhibitor. For instance, and as described by Chambers etal in Endocrinology, 1986, 116:234, incorporated herein by reference,osteoclasts are mechanically disaggregated from neonatal rat long bonesinto Hepes-buffered medium 199. The suspension is agitated, and thelarger fragments are allowed to settle. The cells are then added to twowells of a multi-well dish containing bone slices, and after 15 minutesat 37° C., the bone slices are removed, washed in medium and placed inindividual wells. After 24-hour incubation in 10% FCS in Hanks-bufferedMEM with and without candidate vPPI, the numbers of osteoclasts, and thebone resorption, are quantified by confocal laser scanning microscopy byfixing with 2% glutaraldehyde in cacodylate buffer and staining fortartrate-resistant acid phosphatase (TRAP). After counting the number oflarge multinucleated, red-stained cells, the bone slices are immersed in10% sodium hypochlorite for 60 minutes to remove cells, washed indistilled water and sputter-coated with gold and then re-examined bymicroscopy. The number and size of the osteoclast excavations, the plainarea and the volume of bone resorbed are recorded. By this assay,acidification inhibitors are identified as agents that (1) havesubstantially no effect on osteoclast viability or on the number ofosteoclasts attached to the repair site, and (2) elicit a reduction overcontrol in the mean pit number per osteoclast, and/or mean area perosteoclast and/or mean volume per osteoclast.

Inhibitors of cell-mediated acidification suitable for use in thepresent biomaterials include particularly the so-called proton pumpinhibitors, or PPIs. The PPIs include compounds that are inhibitors ofthe vacuolar proton pump, which is believed to be specific to mammalianosteoclasts. Inhibitors of the vacuolar proton pump, vPPIs, arecharacterized by their ability to inhibit the activity of vATPasepresent in osteoclasts, which in turn interferes with the release ofprotons by “binding” to the subunit C of the vATPase and consequentlysuppresses the creation of the acidic environment in which osteoclastsfunction to degrade bone (see B. Bowman, E. Bowman, J Biol Chem, v.277(6), pp. 3965, 2002). Thus, the present biomaterials may incorporate,as acidification inhibitor, any vPPI that inhibits the activity ofvATPase, and particularly mammalian osteoclast vATPase (also referred toas H⁺-ATPase). The structure of these vATPases is reported for instancein WO93/01280 and by Hall et al in Bone and Mineral Res, 1994, 27:159,the disclosures of which are incorporated herein by reference. ThesevPPIs can be identified using established biological assays, asdescribed in the references just noted, for instance.

Acidification inhibitors useful in the present biomaterials also includeother proton pump inhibitors including many of the macrolideantibiotics. Thus, useful vPPIs include concanamycins A, B and C,analogs thereof including 3′,9-di-O-acetyl concanamycin A, as well asthe corresponding concanolides including concanolides A and B andcertain analogs including 21-deoxy-concanolide A or B,23-O-methyl-concanolide A, 16-demethyl-21-deoxy concanolide A,21,23-dideoxy-23-epiazidoconcanolide A,23-O-(p-nitrobenzenesulfonyl)-21-deoxyconcanolide A, 21-deoxyconcanolideA-23-ketone and the corresponding 9,23-diketone, and21,23-dideoxy-23-epichloroconcanolide A. Other suitable analogs aredescribed in U.S. Pat. No. 5,610,178, incorporated herein by reference.

Also useful as acidification inhibitors are proton pump-inhibitingindole derivatives and heteroaromatic pentadienoic acid derivativesdisclosed in U.S. Pat. No. 5,985,905 and U.S. Pat. No. 6,025,390,respectively, which are incorporated herein by reference.

Useful acidification inhibitors also include particularly thebafilomycins such as A₁, A₂, B₁, B₂, C₁, C₂ and D, the hygrolidinsincluding hygrolidin, hygrolidin amide, defumarylhygrolidin andoxohydrolidin, as well as 16-membered diene macrolides, as described forinstance in U.S. Pat. No. 5,354,773. In embodiments of the presentinvention, the vPPI is either Bafilomycin C₁ or, more preferably,Bafilomycin A₁.

Still other macrolide PPIs and certain non-macrolide PPIs, such aspantoprazole and structurally related benzimidazoles includingomeprazole, may be useful in the present invention provided that theyexhibit proton pump inhibition activity and otherwise meet thebiological criteria noted herein above.

It will be appreciated that the acidification inhibitors can include anycompound that functions to reduce the acidity of the bone remodelingenvironment in which the biomaterial is applied, by acting eitherdirectly or indirectly on the biochemical cascade resulting in protonrelease from the osteoclasts and/or activated macrophages. For instance,in embodiments of the present invention, the acidification inhibitorscan include compounds that act upstream of the pump to inhibit thebiochemical cascade which results in proton release from certain celltypes. Such compounds are identified in established assays such as thatreported for instance by Schlesinger et al in Miner. Electrolyte Metab,1994, 20:31-39, incorporated herein by reference. Briefly, the assayidentifies potential resorption inhibitor compounds that can enter theosteoclast cell through channels other than the proton pump. Suchchannels are found on the osteoclast apical membrane (exposed toextracellular environment) as well as on the ruffled border which isfacing and isolating the osteoclastic lacunae from the extracellularenvironment. The compounds described in this publication were shown toaffect the internal osteoclast pathways that in turn affected thecapability of the cell to acidify the isolated micro-environment in theosteoclast lacunae. Cultured avian osteoclasts were used to test anumber of compounds that could potentially affect the finalacidification pathway. The substrate used for these tests was rat boneparticulate, which was first labeled in vivo with ³H-proline. Theosteoclasts were first allowed to adhere to these labeled bone particlesand bone resorption baseline was established by measuring the release ofthe ³H-proline label (μg) as the cultured osteoclasts were resorbing thebone. The presence of the tracer label did not interfere with theattachment of the osteoclasts to the bone particles as was indicated byactive resorption. Next, the candidate inhibitor compounds were added tothe culture medium and the amount of the released label was measuredagain. A decrease in the amount of the label released was correlatedwith the decrease in resorption, and reveals the candidate inhibitors asinhibitors useful herein as acidification inhibitors.

Included among such acidification inhibitors are chloride pumpinhibitors, such as sumarin, diisothiocyanato-stilbene-disulfonic acid(DIDS), and carbonic anhydrase inhibitors.

Thus, in accordance with the present invention, the resorption of agiven matrix material is controlled biologically by the incorporationtherein of an inhibitor of the osteoclast/macrophage-mediatedacidification process responsible for the in vivo resorption of thatmatrix material.

To provide for the desired resorption control, in accordance with thepresent invention, the selected acidification inhibitor is incorporatedwith the resorbable matrix material in an amount effective, in vivo, toreduce the rate at which the matrix material is resorbed during theremodeling process while maintaining the desired bone tissue healingand/or repair process. The amount of inhibitor effective to confer thisinhibitory activity will vary according to a number of parameters, whichinclude the potency of the compound, the site of biomaterialapplication, the condition being treated and, importantly, the desiredperiod over which the structural integrity of the matrix is required fortreatment.

Experimental results with bafilomycin A1 indicate that there is a PPIconcentration range that is most suitable for achieving control over CAPmatrix resorption. As shown in the examples herein, a dose range studyrevealed that bafilomycin concentrations reach a maximum, beyond whichthey are detrimental to the bone healing process, which likely resultsfrom interference with other cell types following diffusion of theinhibitor outside the implant vicinity. Also, bafilomycin concentrationsbelow a certain threshold fail to show any modulation of the rate atwhich the CAP matrix was resorbed. Moreover, the dose window withinwhich bafilomycin exerts control over CAP matrix degradation does notcorrelate with the far broader dose range suggested as being effectivefor its antibiotic use. Thus, there clearly is a different balancestruck biologically between the effective use of bafilomycin as anantibiotic, and its present use as a means for controlling the rate atwhich matrix resorbs.

In accordance with a particular aspect of the present invention, thereis provided a biomaterial useful to grow, repair and/or heal bone,comprising a CAP matrix material and, as acidification inhibitor, aproton pump inhibitor in an amount effective to control the rate atwhich the matrix is resorbed in vivo.

The amount of a given PPI that would be effective to control in vivoresorption of a given matrix can be determined conveniently using thebiological assay herein exemplified. Briefly, a given CAP matrix isdoped with varying amounts of a given PPI, for instance in the weightrange of from nanograms of PPI to milligrams of PPI per milligram ofmatrix. Sample preparation is achieved by mixing the CAP matrix reagentsin the usual manner, and together with the selected amount of inhibitortypically as a prepared solution. Most suitably, the inhibitor isadmixed first in the liquid phase, before the solid phase of CAPreagents is added, to from the resulting paste. As a PPI-doped paste,the CAP biomaterial can be shaped, while setting, into rods (injectablematerials, lithomorphs, etc.) and then implanted into defects preparedin the femurs of experimental animals. After a desired treatment period,the femurs are removed; histological sections thereof prepared, and thenexamined under microscopy for implant degradation, and for the presenceof typical osteoclast colonies in the implant zone. Effectiveamounts/concentrations of a given PPI are revealed as thoseamounts/concentrations that are useful in combination with matrixmaterial for increased growth, healing and/or repair of bone tissue, andthat elicit a reduction, relative to un-doped control, in thedeterioration of the matrix. In biological terms, the effective doserange for a given acidification inhibitor is revealed as a minimumeffective to permit but reduce the number of vicinal cutting cones andthe incursion of resorptive cells into the matrix, and a maximumeffective to maintain increased growth, healing and/or repair process,i.e., to permit any initially formed fibrous tissue or fibrous capsuleto resolve and to limit the recruitment of inflammatory cells to thematrix application site.

In one embodiment, the present invention provides a CAP biomaterialcomprising a near-ambient CAP matrix, and bafilomycin A1 in an amounteffective to reduce the rate at which said matrix is degraded in vivo.In particular embodiments, the near ambient CAP matrix comprisesdicalcium phosphate and tetracalcium phosphate. In other embodiments,the bafilomycin A1 is present in an amount of from 0.5-6.5×10(−4) M, forexample of from 1.0-3.0×10(−4) M, and particularly about 1.4-1.8×10(−4)M in the liquid phase used to prepare the cements. Expressed in weightamounts, the bafilomycin A1 is suitably incorporated in amounts rangingfrom about 25-100 μg, desirably 30-70 μg, more desirably 35-65 μg andmost desirably 40-60 μg in healthy and osteopenic rats per 500 mg CAPmatrix, e.g., about 40 μg per 500 mg of near ambient CAP matrix.Expressed as weight ratios, the bafilomycin A1:CAP matrix ratio (mgPPI:mg CAP) lies desirably within the range from about 1:20,000 to about1:5,000, desirably 1:17,000 to 1:7,000, more desirably 1:15,000 to1:7,500, and most desirably 1:12,500 to 1:8,000.

It will be appreciated that the specific dose of acidification inhibitoruseful to control matrix resorption will depend of course on the potencyof the particular inhibitor, on the type of matrix material selected,and on the technique used to produce the inhibitor-doped matrixmaterial. It will also be appreciated that the assays described hereintogether with the results provided herein for the bafilomycin/CAPcombination and the other noted combinations with pantoprazole and withCalcibon® will provide guidance sufficient to determine optimumparameters for the production of other biomaterials that function on thesame principle.

The PPI-doped CAP biomaterials can be prepared by applying those methodsalready established for CAP biomaterials, but with the additional stepof incorporating the PPI into the CAP matrix at any convenient point inthe process. Suitably, the PPI is sufficiently soluble in the liquidphase of the CAP reagents, and is introduced into this phase, in thedesired amount, before the final paste is formed. Alternatively, the PPIcan be added, with mixing, to the solid phase reagents, and thendissolved therewith into the liquid reagent phase. As a furtheralternative, the PPI can be used as a coating on a pre-formed CAPmatrix, and can be applied by spraying onto the matrix surface or bydipping the matrix into a solution thereof to impregnate the poreswithin the matrix. Thus, it will also be appreciated that the PPI can beused as dopant for a wide variety of CAP matrix materials that allow forthe PPI to elute or leach from the matrix during its resorption orbiodegradation, or allow the PPI to remain associated with the matrixfor encounter with incursive osteoclasts. Suitably, the CAP matrix is anear-ambient CAP matrix, having the advantage of setting at near ambienttemperature, and setting with minimal exothermic energy.

Similarly, the acidification inhibitor can be incorporated into othermatrix materials including the PLGAs. For instance, the PLGAs can beformed in two dimensions by curing on a flat surface. The resultingtwo-dimensional polymer can then be doped with the acidificationinhibitor simply by immersing the polymer in a solution containing theselected acidification inhibitor, or by spraying the surface of thepolymer with that solution, to provide a desired dose of the inhibitoron the osteoclast contact surface thereof For instance, the stepsrequired for the preparation of such surface involve dissolving thepolymer such as PLGA (e.g., PLGA 75:25, inherent viscosity 0.87 dL/g,Birmingham Polymer Inc., Alabama) in a volatile solvent such aschloroform at 2% (w/v) for example. The viscosity of the polymersolution can be adjusted as needed. The PPI can either be dissolved inan appropriate solvent and incorporated into the polymer solution, orthe PPI can be dissolved in the initial solvent to be used fordissolving the polymer pellets.

To create the 2-dimensional polymer-inhibitor surfaces, the resultantpolymer-inhibitor solution is applied to sterile glass coverslips(Bellco, N.J.) using a procedure called spin coating. While thecoverslips are being spun at 5500 rpm using a photolithographic spinner(Headway research Inc., Texas), 0.5 mL of the 2% polymer-inhibitorsolution is applied drop-wise to the spinning coverslip over a120-second period using a pipette. After spin coating, the coverslipsare air-dried and rinsed with α-minimal essential medium, α-MEM, (a cellfeeding solution). The control polymer surfaces should be prepared insimilar fashion but not contain the inhibitor.

The resultant polymer-inhibitor coated coverslips and the controlcoverslips can also be used for osteoclastic cell culture as described,for example, by Davies et al. Polymer-inhibitor coated coverslips shouldalso be immersed in the tissue culture medium without the osteoclastsand used as negative controls for polymer degradation and inhibitordissolution from polymer matrix.

Furthermore, the polymer can dissolved in a suitable solvent, e.g. PLGAin chloroform, and the PPI (in solid or liquid form) can be incorporatedinto the polymer solution, which can subsequently be administered as aliquid or allowed to harden and used as a solid implant.

As with the CAP matrix per se, the present biomaterials can be utilizedin wide variety of clinical settings, to induce the growth, healing andrepair of bone tissues in various anatomical sites. Such end-usesinclude, by are by no means limited to, dental applications, fracturerepair, arthroplasty, cranio-facial plastic reconstruction, sinus liftfiller, and vertebroplasty, and for the local treatment of theseconditions secondary to such bone diseases and conditions as osteopenia,osteoporosis, and the like. The present CAP biomaterials provide theadditional advantage that, with the addition of effective PPI amounts,the CAP matrix can be retained for treatment periods that are morehighly controlled and more appropriate for the desired therapy. It willfurther be appreciated that biomaterials based on antibiotic PPIs canusefully be applied to bone sites at which infection is present, therebytaking advantage of the antibiotic properties of the PPI presenttherein. However, it will also be appreciated that the same biomaterialis usefully applied regardless of the infection status of theapplication site, and can effectively be used at sites at whichinfection or biotic contamination is not present.

It will also be appreciated that the present biomaterials can furtherincorporate other ingredients that might usefully be delivered to a bonesite, to promote healing and the like, such as bone growth factors,organic polymers such as fibrin, and the like.

EXAMPLE 1 Materials and Methods Chemicals

Dicalcium phosphate anhydrous (CaHPO₄, DCPA) and Bafilomycin A₁,dimethyl sulfoxide (DMSO) and methanol (MeOH) were purchased fromSigma-Aldrich Inc. (Oakville, ON, Canada). Tetracalcium phosphate(Ca₄(PO₄)₂O, TTCP) was purchased from Clarkson Chromatography ProductsInc. (South Williamsport, Pa., USA). Compounds used for the preparationof the liquid phase, disodium hydrogen phosphate (Na₂HPO₄) and sodiumdihydrogen phosphate (NaH₂PO₄), were purchased from BDH Inc. (Toronto,ON, Canada).

Preparation of CAP Pastes and Pre-set Cement Rods

The calcium phosphate precursor powder consisted of an equimolar mixtureof DCPA and TTCP sterilized by gamma irradiation (20 kGy). Deionizeddouble distilled water was obtained from a Millipore Milli-RO 10 Plusand Mill-Q UF Plus systems (Bedford, Mass., USA) operated at 18 MΩresistance. The liquid phase was a neutral sodium hydrogen phosphatesolution at pH 7.4, which was sterilized by filtration through 0.22 μmfilters (Millipore Corp., Bedford, Mass., USA).

Bafilomycin A₁ was first dissolved in DMSO solution (other solutionsthat can be used are methanol and ethanol), and then dispersed in aneutral phosphate solution (pH 7.4) prepared from Na₂HPO₄ and NaH₂PO₄.

Three concentrations of Bafilomycin A₁ were investigated: 1.61×10⁻⁵ M(DMSO solvent), 1.61×10⁻⁴ M (DMSO solvent), and 2.0×10⁻³ M (methanolsolvent was used because the solubility of Bafilomycin A₁ in DMSO wasexceeded).

Cements with the lowest concentration did not differ in histology(resorption occurred in both control and the cement with BafilomycinA₁). The highest concentration did not work either (the bone would notheal around the implant).

Concentration of 1.61×10⁻⁴ M was found to work very well, and thereforewas chosen to be the standard concentration for all of the experiments.

After Bafilomycin A₁ was dispersed in the biphasic liquid phase, theliquid phase was added to one of the solid components of the CAP,dicalcium phosphate anhydrous (DCPA). The DCPA was dissolved in theliquid phase and left at room temperature for 2 min with continuousmixing.

Next, tetracalcium phosphate (TTCP) was gradually added to the firstdissolved CAP component to produce a paste. The powder to liquid ratio(P/L) of the final CAP was 2.0 (wt/wt).

The pastes were either implanted or used for the preparation of rods.Cement rods (1.9 mm diameter×2.3 mm height) were shaped by allowingaliquots of packed CPC pastes to set in custom made Teflon® formers for24 h at 37C and 100% humidity and subsequently for 48 h at roomtemperature.

Implantation Procedure

Young male Wistar rats with an average body weight of 125-150 g werepurchased from Charles River (QC, Canada), housed in light- andtemperature-controlled rooms, and fed a standard diet. The maintenanceand use of animals were in accordance with the Canadian Council ofAnimal Care Guidelines.

Bone defects were drilled in the mid-diaphysis of the femur. The holeswere made using a low speed dental drill (2.3 mm in diameter) withcopious saline irrigation. Pre-hardened CPC rods were placed into theholes using gentle pressure. Every rat received two implants, a controlrod (paste) in one femur and a rod (paste) containing Bafilomycin A₁ inanother femur. The animals were observed daily throughout theimplantation period.

Histological Processing

After desired time in vivo, the animals were sacrificed, and the femurswere removed. The femurs were then exposed to a fixative solution for 48h, decalcified in a 1:1 mixture of 45% formic acid and 20% sodiumcitrate, dehydrated and embedded in low-melting-point paraffin. Serialsections (6 μm thick) perpendicular to the long axis of the implant wereobtained using a hard tissue Spencer 820 microtome. Sample sections werestained with hematoxylin-eosin (H&E) and Mason's trichrome stains.Representative sections were also tested for the presence of tartrateresistant acid phosphatase (TRAP) enzyme, characteristic of osteoclasts,to determine the identity of the cells at the material/bone interface.

Results

A. Appearance of the Implanted Cement Rods and the Bone-materialInterface

FIGS. 1A shows that the initially cylindrical control rod becamedistorted and irregular around the periphery. Closer examination of theperipheral surface and the bulk matrix of the control cement revealed ahigh degree of cellular activity (FIG. 2A). The osteoclastic “cuttingcones”, characteristic of resorption, were apparent. The cells at thetips of these “cutting cones” were large, multinucleated, and irregularin morphology.

In contrast to the controls, the cement rods containing Bafilomycin A₁were not distorted and had a smooth periphery (FIG. 1B). Large,elongated and multinucleated cells surrounded the periphery of the rod(FIG. 2B).

B. New Bone Surrounding the Implants

A layer of new bone surrounded both the control and the cement modifiedwith Bafilomycin A₁ (FIG. 1). The amount of new bone was lower in thecontrols than in the Bafilomycin A₁-modified cements, which suggeststhat the rates of remodeling varied. The CAP materials, which wereimplanted as pastes showed the same response as that observed for thepre-set cements (FIG. 4).

As is demonstrated by the histology (FIGS. 1, 2, 4 & 5), presence ofBafilomycin A₁ in the CAP cements decreased the resorption rate of thesematerials in comparison to the controls, which did not contain thisvacuolar proton pump inhibitor. The pre-set cement rods containingBafilomycin A₁ retained their original cylindrical shape throughout theimplantation period studied. In contrast, the appearance of the controlcement rods was somewhat distorted, which can be attributed to the highdegree of cellular activity (osteoclastic resorption) at the edges anddeeper in the material matrix. In the control cements, the “cuttingcones”, which are typically seen in metabolically active bone, werecutting into the matrix of the rods. The morphology of themultinucleated cells at the forefront of the “cutting cones” wascharacteristic of the osteoclasts, which are responsible for resorbingbone. These multinucleated cells showed positive staining for theenzyme, TRAP, (FIG. 3) which confirmed their identity to be osteoclasts.The cements containing Bafilomycin A₁ did not show the typicalresorption pattern described above. The multinucleated cells, which werelined-up around the periphery of the material, were elongated and flat.Even though these cells did not form “cutting cones”, they didpositively stain for TRAP, which again identified them as osteoclasts.These osteoclasts, however, were not resorbing the material, becausetheir proton pumps were inhibited, and therefore the original shape ofthe material was retained.

In all of the specimens studied, the cements containing Bafilomycin A₁were consistently surrounded by more bone than the control cements. Newbone surrounding the Bafilomycin A₁ cements appeared to be denser thanthe bone around the controls, which can be attributed to the decreasedresorptive activity of the osteoclasts. The higher amount of bonesurrounding the CPC containing Bafilomycin A₁ is considered a beneficial“side effect” elicited by the vacuolar pump inhibitor. Bafilomycin A₁not only slowed down resorption of the material, but it also slowed downresorption of the newly formed bone around the cement by leaching intothe peri-implant environment.

It will thus be appreciated that resorption of near-ambient temperatureCAP materials can be biologically controlled by introducing a vacuolarproton pump inhibitor into the cement. Bafilomycin A₁ inhibited CAPcement resorption by interfering with the function of the vacuolarproton pumps located at the ruffled border of osteoclasts, which wouldotherwise resorb the material.

EXAMPLE 2

Performance of the bafilomycin-doped implant was also assessed in amodel of osteoporosis which utilizes ovarectomized aged rats. Successfulintegration of osseous implants with host bone tissue can be adverselyaffected by pathological bone conditions such as osteoporosis, whichresult in osteopenia due to disturbance of the balance between boneresorption and bone formation processes. Therefore, systemic bone lossdue to estrogen deficiency may also affect bone growth and maintenancearound the implants. Although current attention has been focused in theart on systemic fracture prevention and development of new therapiesaimed at conserving bone mass, little emphasis has been given toincreasing osteointegration of implants in osteoporotic defect site.

The effects of ovariectomy on bone tissue formation and resorption ofproton pump inhibitor (Bafilomycin A₁)—modified resorbable CAP cementimplants in comparison to the controls were studied.

Implantation Procedure

Ten-month old ovariectomized (ovx) virgin Brown Norway rats with werepurchased from the National Institute on Aging (Bethesda, Md., USA),housed individually in light- and temperature-controlled rooms, and feda restricted diet (6 pellets/day). These animals were kept foradditional four months following the ovariectomy. Non-ovx virgin BrownNorway rats of the same age as the experimental rats at the time ofimplantation (fourteen months) were used as a control group for the ovxanimals. Young virgin female Brown Norway rats (three months old) wereused as another comparison group to assess the implant integration andresorption. The maintenance and use of animals were in accordance withthe Canadian Council of Animal Care Guidelines.

Materials and Methods

Production of the CAP implant doped with bafilomycin was performed asdescribed above, i.e., resorbable CAP based on the TTCP and DCPAchemistry. Bafilomycin in concentration of 75-100 μg per 250 μL ofliquid neutral phosphate phase per 500 mg of CAP was used. The controldid not contain the proton pump inhibitor.

The implants were allowed to set to form pre-hardened implant rods. Theimplants were retrieved at 10 days, 1 month, 2 months, and 4 monthspost-surgery.

Results

FIGS. 6 and 7 show sample results for short-period implantation (10days) and long-term implantation (4 months) respectively obtained forthe ovx aged rats. Trends in results obtained for the young controlgroup and the non-ovx aged group were similar with respect to trends ofreduced resorption of the materials doped with PPI and increased boneformation around the material doped with PPI, which were observed evenat 4 months in vivo.

These results indicate that the PPI entrapped in the CAP matrix appearsto be active throughout the 4-month in vivo period as is demonstrated bythe reduction of the resorption of the experimental sample in comparisonto the control material. Furthermore, these results also indicate thatCAP implant doped with a PPI can be used to allow more effectiveincorporation of the implant in a compromised bone tissue environment asa result of increased local bone mass.

EXAMPLE 3

Experiments with a commercially available proton pump inhibitor, sodiumpantoprazole sold as Panto IV®, were performed in healthy young Wistarrats.

Panto IV,5-difluoromethoxy-2-[(3,4-dimethoxy-2-pyridinyl)methyl]-1H-benzimidazole,belongs to the chemical family of substituted benzimidazoles, which alsoincludes omeprazole (Losec®), and lansoprazole (Prevacid®), and is usedclinically to treat peptic ulcers. Compounds of this class exist aspro-drugs that need to be activated by the acidic pH to form activesulphenamides before they can interact with the gastric H⁺,K⁺-ATPase.The compounds are chemically stable at neutral pH, and when they reachthe parietal cells (stomach), protonation of these compounds results inmolecular rearrangement followed by the formation of an activesulfonamide compound. The activated compound then reacts covalently withthe sulfhydryl groups on the surface of the H⁺,K⁺-ATPase.

Although Panto IV is highly specific for the proton pumps of theparietal cells, it was hypothesized that it may non-specifically affectother proton pumps, including the osteoclast proton pump. A commerciallyavailable calcium phosphate material, Calcibon, was also used which isless resorbable than the previously used CAP to determine whether thestrategy for stimulating local bone mass can be used with othermaterials.

Materials and Methods

The CAP material used was Calcibon provided by the MerckBiomaterials/Biomet company. The concentration used was 4 mg Panto IVper/mL of neutral phosphate solution per 500 mg of CAP powder used toprepare the cement. The cement doped with the PPI was then allowed toset, as described previously. The pre-set rods were implanted in youngWistar rats according to the same procedure as described previously.

Results

Calcibon is not a readily resorbable calcium phosphate cement, thereforeno resorption of the cement rod at 10 days in vivo was observed, asexpected. However, as shown in FIG. 8, the experimental sample wassurrounded by higher reparative bone mass than the control, which isconsistent with the trends observed when a macrolide PPI was used.Because Panto IV is specific for the H⁺, K⁺-ATPase and not for theosteoclast proton pump, a higher dose was required in order to achieveresults similar to those obtained with Bafilomycin A₁, which is aspecific inhibitor of the osteoclast proton pump. This exampledemonstrates that the present invention can be applied with proton pumpinhibiting compounds other than those from the class of macrolideantibiotics.

It will thus be appreciated that implants formed of bone compatiblematrix materials can usefully be doped to provide control over the rateat which they are biodegraded following implantation, by incorporatingan inhibitor of cell-mediated acidification of the bone remodelingenvironment. Suitable inhibitors include the proton pump inhibitors, andparticularly inhibitors of the osteoclast proton pump. Using thesedopants, matrix materials that normally are degraded too rapidly to beuseful in a given bone healing environment can be rendered more suitableto that application, to provide a slower rate of degradation, byincorporating the acidification inhibitor. Moreover, by providingcontrol over the rate of matrix degradation, the acidification inhibitorin effect slows the rate of its own release from that matrix, and thusprovides for longer term control over the acidification environment. Inaddition, by incorporating the inhibitor, the present implants alsoprovide the additional medical benefit that new bone formation ispromoted in the region exposed to the acidification inhibitor.

1. A biomaterial of the type suitable, when implanted into a local boneremodeling environment, to promote the localized growth and/or repair ofbone tissue, the biomaterial comprising a bone compatible matrixmaterial and an inhibitor of cell-mediated acidification in an amounteffective to reduce cell-medicated acidification in the bone remodelingenvironment in which the biomaterial is implanted.
 2. A biomaterialaccording to claim 1, wherein the inhibitor of cell-mediatedacidification is present in an amount effective to promote the growth ofnew bone.
 3. A biomaterial of the type suitable for use in localizedbone healing and/or repair, the biomaterial comprising a resorbable bonecompatible matrix material and an inhibitor of cell-mediatedacidification in an amount effective to reduce the rate at which saidmatrix is resorbed in vivo.
 4. A biomaterial according to claim 1,wherein the resorbable matrix material is a near-ambient calciumphosphate matrix material.
 5. A biomaterial according to claim 1,wherein the inhibitor of cell-mediated acidification is a proton pumpinhibitor.
 6. A biomaterial according to claim 1, wherein the calciumphosphate matrix material is calcium phosphate cement.
 7. A biomaterialaccording to claim 1, wherein the proton pump inhibitor is amacrolide-based antibiotic.
 8. A biomaterial according to claim 1,wherein the proton pump inhibitor is bafilomycin Al.
 9. A biomaterialaccording to claim 8, wherein the bafilomycin is present in thebiomaterial at a concentration within the range from 25-100 μg per 500mg of matrix.
 10. A biomaterial according to claim 1, in the form ofshaped, hardened agent or paste.
 11. A method for treating a subject toinduce the growth or repair of bone tissue, the method comprising thestep of implanting, at the site at which bone growth or repair isdesired, a biomaterial according to claim
 1. 12. The method according toclaim 11 for the treatment of a subject to induce the growth of bonetissue in a local bone environment, the method comprising the steps ofapplying, to the site at which bone growth is desired, said biomaterial.13. The method according to claim 11 for the treatment of a. subject toinduce the repair of bone tissue in a local bone environment, the methodcomprising the steps of applying, to the site at which bone repair isdesired, said biomaterial.
 14. A method according to claim 11, whereinthe biomaterial is applied to a site at which infection is absent. 15.The use of a biomaterial according to claim 1 to promote the localizedgrowth and/or repair of bone tissue.
 16. The use according to claim 15,for the repair of bone fracture.
 17. The use according to claim 15, forthe repair of bone grafts.
 18. The use according to claim 15, for thetreatment of osteoporosis.
 19. A process for preparing a biomaterial ofthe type useful to induce the growth and/or repair of bone tissue, theprocess comprising the steps of combining reagents capable of forming arestorable matrix material, and inhibitor of cell-mediated acidificationin an amount effective to control the rate at which said matrix materialis resorbed in vivo.
 20. A process for preparing a biomaterial of thetype useful to induce the growth and/or repair of bone tissue,comprising the step of combining a settable calcium phosphate cementwith bafilomycin A1 in a dose effective to reduce cell-mediatedacidification in a bone remodeling environment in which the biomaterialis implanted.